Biochemical and structural investigation of mutations in Eg5 that confer resistance to anti-mitotic drugs


Session type:

Sandeep Kumar Talapatra1, Venkatasubramanian Ulaganathan1, Frank Kozielski1
1The Beatson Institute for Cancer Research, Glasgow, Scotland, United Kingdom


The mitotic kinesin Eg5 is essential for the separation of duplicated centrosomes and bipolar spindle formation in dividing cells. Eg5 is a novel anti-cancer target, which is thought to overcome the drawbacks of microtubule targeting drugs. Various structurally diverse small molecules, such as Ispinesib, STLC and others have been found to inhibit Eg5. They target an allosteric inhibitor binding pocket consisting of loop L5, helix ?2 and ?3 in the Eg5 motor domain. However, certain mutations in the loop L5 region can render these drugs ineffective.

We investigated Ispinesib resistant Eg5 mutants to understand how they reduce efficacy of Eg5 targeting drugs.


Wild-type and mutated Eg5 were cloned, expressed and purified. In vitro ATPase assays were used to assess the kinetics of the wild-type and mutants in the presence of a variety of Eg5 targeting drugs, including compounds in Phase I and Phase II clinical trials. In addition, we have also determined the x-ray crystal structures of several of these resistant Eg5 mutants.


In vitro ATPase activity assays (both basal and microtubule stimulated) suggest that Ispinesib and STLC like compounds are potent inhibitors of Eg5. However, mutations in the allosteric inhibitor-binding pocket substantially reduce the potency of the drugs. We have solved crystal structures of these mutants to understand the structural basis of resistance. Compared to the native Eg5 structure, the mutants display only minor structural differences.


The mutations don’t cause major conformational changes in Eg5. The change in the side chain of the mutated residue causes steric hindrance in the inhibitor binding pocket which in turn reduces the potency of Eg5 targeting drugs. Further work will involve designing new inhibitors to overcome the interference caused by mutations in the inhibitor-binding pocket.